Bile salt hydrolase of Bifidobacterium longum-biochemical and genetic characterization

Abstract

A bile salt hydrolase (BSH) was isolated from Bifidobacterium longum SBT2928, purified, and characterized. Furthermore, we describe for the first time cloning and analysis of the gene encoding BSH (bsh) in a member of the genus Bifidobacterium. The enzyme has a native molecular weight of 125,000 to 130,000 and a subunit molecular weight of 35,024, as determined from the deduced amino acid sequence, indicating that the enzyme is a tetramer. The pH optimum of B. longum BSH is between 5 and 7, and the temperature optimum is 40 degrees C. The enzyme is strongly inhibited by thiol enzyme inhibitors, indicating that a Cys residue is likely to be involved in the catalytic reaction. The BSH of B. longum can hydrolyze all six major human bile salts and at least two animal bile salts. A slight preference for glycine-conjugated bile acids was detected based on both the specificity and the K(m) values. The nucleotide sequence of bsh was determined and used for homology studies, transcript analysis, and construction and analysis of various mutants. The levels of homology with BSH of other bacteria and with penicillin V acylase (PVA) of Bacillus sphaericus were high. On the basis of the similarity of BSH and PVA, whose crystal structure has been elucidated, BSH can be classified as an N-terminal nucleophile hydrolase with Cys as the N-terminal amino acid. This classification was confirmed by the fact that a Cys1Ala exchange by site-directed mutagenesis resulted in an inactive protein. Reverse transcription-PCR experiments revealed that bsh is part of an operon containing at least two genes, bsh and glnE (GlnE is glutamine synthetase adenylyltransferase). Two UV-induced BSH-negative mutants and one spontaneous BSH-negative mutant were isolated from B. longum SBT2928 cultures and characterized. These mutants had point mutations that inactivated bsh by premature termination, frameshift, or amino acid exchange.

FIG. 1

Genetic and transcriptional organization of the bsh site (A) and RT-PCR analysis of the transcript of the bsh region (B and C). P_bsh, putative bsh promoter; A through E, primers A through E used for RT-PCR (Table 2); MWM, molecular weight marker; DNA, control reaction; +AMV, first-strand reaction with AMV; −AMV, first-strand reaction without AMV. (B) First-strand cDNA synthesis with primer E. (C) First-strand cDNA synthesis with primer C. In the lanes in which primers A and C were used and in the lanes in which primers A and F were used three times as much RT-PCR product was applied in order to obtain bands whose intensity was similar to that of the DNA control.

FIG. 2

Purification and immunostaining of B. longum BSH. (A) SDS-PAGE (10% polyacrylamide) after silver staining. Lane MW, molecular weight marker; lane CFE, cell extract; lanes Q, S, and GF, BSH active fraction after MonoQ chromatography, MonoS chromatography, and gel filtration, respectively. (B) Immunostaining with anti-C. perfringens BSH antibodies. Lane CFE, cell extract; lanes Q, S, and GF, BSH active fraction after MonoQ chromatography, MonoS chromatography, and gel filtration, respectively; lanes P and N, cell extract of E. coli with and without C. perfringens BSH, respectively.

FIG. 3

Alignment of BSH of various bacteria, of a hypothetical protein of Bacillus subtilis, and PVA of Bacillus sphaericus. Asterisks, identical amino acids; dots, similar amino acids. The boxes indicate amino acids involved in the active site of PVA, including C-1, D-20, Y-82, N-175, and R-228 (positions based on the B. sphaericus DNA sequence). The amino acid sequence which was determined with purified BSH is indicated by boldface type. The enzymes examined were the BSH of B. longum (BSHBLO), L. johnsonii BSH (BSHLBJ), L. plantarum (BSHLBP), and C. perfringens (BSHCLP); the hypothetical protein of Bacillus subtilis (HYPBSU); and Bacillus sphaericus PVA (PENABSP).

FIG. 4

Plasmid maps of plasmids pBH13 and pBH16, which confer BSH activity, and two subclones. Heavy lines, plasmid pUK21; boldface restriction sites, sites present in the insert; P_lac, E. coli lacZ promoter.

FIG. 5

Nucleotide sequence of the bsh gene region of B. longum. IR, inverted repeat; DR, direct repeat; −10, putative −10 promoter region; SD, putative SD sequence. The N-terminal amino acid sequence determined for purified BSH is underlined. Mutations are indicated above the nucleotide sequence by boldface letters which indicate the exchanged or inserted nucleotides. The amino acids of the supposed active site (C-1, D-20, N-81, N-172, and R-225) are double underlined.